Fractionation of charged keratin

ABSTRACT

Methods are provided to produce optimal fractionations of charged keratins that have superior biomedical activity. Also provided are medical implants coated with these keratin preparations. Further provided are methods of treating blood coagulation in a patient in need thereof.

RELATED APPLICATIONS

This application is a divisional of and claims priority to U.S. patentapplication Ser. No. 12/279,641, filed Sep. 2, 2008, which is a nationalphase application of PCT Application PCT/US2007/004193, filed Feb. 15,2007, and published in English on Aug. 30, 2007, as InternationalPublication No. WO 2007/098053, and which claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Patent Application Ser. No.60/774,442, filed Feb. 17, 2006, U.S. Provisional Patent ApplicationSer. No. 60/774,587, filed Feb. 17, 2006, and U.S. Provisional PatentApplication Ser. No. 60/774,920, filed Feb. 17, 2006, the disclosures ofeach of which is incorporated herein by reference in its entirety.

This application is related to: Mark E. Van Dyke, U.S. patentapplication Ser. No. 11/205,800, titled: Ambient Stored Blood PlasmaExpanders, filed Aug. 17, 2005, and published on Mar. 9, 2006, as2006/0051732; Mark E. Van Dyke, U.S. patent application Ser. No.11/673,212, titled: Nerve Regeneration Employing Keratin Biomaterials,filed Feb. 9, 2007, and published on Dec. 27, 2007, as 2007/0298070; andMark E. Van Dyke, U.S. patent application Ser. No. 11/676,072, and PCTApplication (published Aug. 30, 2007 as WO2007/098114), titled: Clottingand Healing Compositions Containing Keratin Biomaterials, filed Feb. 16,2007.

GOVERNMENT SUPPORT

This invention was made with Government support under contract numberW81XWH-04-1-0105 from the United States Army. The U.S. Government hascertain rights to this invention.

FIELD OF THE INVENTION

The present invention is generally related to keratin biomaterials andthe use thereof in biomedical applications.

BACKGROUND OF THE INVENTION

The earliest documented use of keratin in medicine comes from a Chineseherbalist named Li Shi-Zhen (Ben Cao Gang Mu. Materia Medica, adictionary of Chinese herbs, written by Li Shi Zhen (1518-1593)). Over a38-year period, he wrote a collection of 800 books known as the Ben CaoGang Mu. These books were published in 1596, three years after hisdeath. Among the more than 11,000 prescriptions described in thesevolumes, is a substance known as Xue Yu Tan, also known as CrinisCarbonisatus, that is made up of ground ash from pyrolized human hair.The stated indications for Xue Yu Tan were accelerated wound healing andblood clotting.

In the early 1800s, when proteins were still being called albuminoids(albumin was a well known protein at that time), many different kinds ofproteins were being discovered. Around 1849, the word “keratin” appearsin the literature to describe the material that made up hard tissuessuch as animal horns and hooves (keratin comes from the Greek “kera”meaning horn). This new protein intrigued scientists because it did notbehave like other proteins. For example, the normal methods used fordissolving proteins were ineffective with keratin. Although methods suchas burning and grinding had been known for some time, many scientistsand inventors were more interested in dissolving hair and horns in orderto make better products.

The resolution to this insolubility problem came from a trade more than700 years old—the tanning industry. In the years preceding World War I,lime was applied to the manufacture of keratin gels. In a United Statespatent issued in 1905, John Hoffmeier described a process for extractingkeratins from animal horns using lime (German Pat No. 184,915, Dec. 18,1905). He then used the extracted keratins to make gels that could bestrengthened by adding formaldehyde (formaldehyde “crosslinking” is apopular method of strengthening such gels and is still used today to“fix” tissues containing structural proteins like keratin and collagen).

During the years from 1905 to 1935, many methods were developed toextract keratins using oxidative and reductive chemistries (Breinl F andBaudisch O, Z physiol Chem 1907; 52:158-69; Neuberg C, U.S. Pat. No.926,999, Jul. 6, 1909; Lissizin T, Biochem Bull 1915; 4:18-23; Zdenko S,Z physiol Chem 1924; 136:160-72; Lissizin T, Z physiol Chem 1928;173:309-11). By the late 1920s many techniques had been developed forbreaking down the structures of hair, horns, and hooves, but scientistswere confused by the behavior of some of these purified proteins.Scientists soon concluded that many different forms of keratin werepresent in these extracts, and that the hair fiber must be a complexstructure, not simply a strand of protein. In 1934, a key research paperwas published that described different types of keratins, distinguishedprimarily by having different molecular weights (Goddard D R andMichaelis L, J Biol Chem 1934; 106:605-14). This seminal paperdemonstrated that there were many different keratin homologs, and thateach played a different role in the structure and function of the hairfollicle.

It was during the years of World War II and immediately after that oneof the most comprehensive research projects on the structure andchemistry of hair fibers was undertaken. Driven by the commercializationof synthetic fibers such as Nylon and polyester, Australian scientistswere charged with protecting the country's huge wool industry. Syntheticfibers were seen as a threat to Australia's dominance in woolproduction, and the Council for Scientific and Industrial Research(later the Commonwealth Scientific and Industrial Research Organisationor CSIRO) established the Division of Protein Chemistry in 1940. Thegoal of this fundamental research was to better understand the structureand chemistry of fibers so that the potential applications of wool andkeratins could be expanded.

CSIRO scientists developed many methods for the extraction, separation,and identification of keratins. In 1965, CSIRO scientist W. GordonCrewther and his colleagues published the definitive text on thechemistry of keratins (Crewther W G et al., The Chemistry of Keratins.Anfinsen C B Jr et al., editors. Advances in Protein Chemistry 1965.Academic Press. New York: 191-346). This chapter in Advances in ProteinChemistry contained references to more than 640 published studies onkeratins. Once scientists knew how to extract keratins from hair fibers,purify and characterize them, the number of derivative materials thatcould be produced with keratins grew exponentially. In the decadebeginning in 1970, methods to form extracted keratins into powders,films, gels, coatings, fibers, and foams were being developed andpublished by several research groups throughout the world (Anker C A,U.S. Pat. No. 3,642,498, Feb. 15, 1972; Kawano Y and Okamoto S, KagakuTo Seibutsu 1975; 13(5):291-223; Okamoto S, Nippon Shokuhin KogyoGakkaishi 1977; 24(1):40-50). All of these methods made use of theoxidative and reductive chemistries developed decades earlier.

In 1982, Japanese scientists published the first study describing theuse of a keratin coating on vascular grafts as a way to eliminate bloodclotting (Noishiki Y et al., Kobunshi Ronbunshu 1982; 39(4):221-7), aswell as experiments on the biocompatibility of keratins (Ito H et al.,Kobunshi Ronbunshu 1982; 39(4):249-56). Soon thereafter in 1985, tworesearchers from the UK published a review article speculating on theprospect of using keratin as the building block for new biomaterialsdevelopment (Jarman T and Light J, World Biotech Rep 1985; 1:505-12). In1992, the development and testing of a host of keratin-basedbiomaterials was the subject of a doctoral thesis for French graduatestudent Isabelle Valherie (Valherie I and Gagnieu C. Chemicalmodifications of keratins: Preparation of biomaterials and study oftheir physical, physiochemical and biological properties. Doctoralthesis. Inst Natl Sci Appl Lyon, France 1992). Soon thereafter, Japanesescientists published a commentary in 1993 on the prominent positionkeratins could take at the forefront of biomaterials development(Various Authors, Kogyo Zairyo 1993; 41(15) Special issue 2:106-9).

Taken together, the aforementioned body of published work isillustrative of the unique chemical, physical, and biological propertiesof keratins. However, there remains a need to create optimalfractionations of keratins that have superior biomedical activity.

SUMMARY OF THE INVENTION

The invention provides methods of making charged (i.e. acidic and basic)keratins by separating one from the other, e.g., by chromatography, andoptionally further processing or purifying the retained fraction orfractions. In some embodiments, the keratins fractionated based onacidity consist essentially of alpha keratoses, gamma keratoses, ormixtures thereof. In other embodiments, the keratins fractionatedconsist essentially of alpha kerateines, gamma kerateines, or mixturesthereof.

Another aspect of the present invention is an implantable biomedicaldevice, comprising: a substrate and a keratin derivative on thesubstrate, wherein the keratin derivative is present in an amounteffective to reduce cell and tissue adhesion to the substrate. In someembodiments the keratin derivative comprises, consists of or consistsessentially of basic alpha keratose, basic gamma keratose, basic alphakerateine, basic gamma kerateine, or combinations thereof.

A further aspect of the present invention is an implantableanti-adhesive tissue barrier, comprising: a solid, physiologicallyacceptable substrate; and a keratin derivative on the substrate. In someembodiments the keratin derivative comprises, consists of or consistsessentially of basic alpha keratose, basic gamma keratose, basic alphakerateine, basic gamma kerateine, or combinations thereof.

Yet another aspect of the present invention is a method of treatingblood coagulation in a subject in need thereof, comprising administeringa keratin derivative to said subject in an amount effective to inhibitblood coagulation in said subject, wherein said keratin derivativeconsists essentially of basic keratose, basic kerateine, or combinationsthereof.

Another aspect of the present invention is the use of a keratinderivative as described herein for the preparation of a composition ormedicament for carrying out a method of treatment as described herein,or for making an article of manufacture as described herein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The unique properties of subfamilies of keratins can be revealed andutilized through more sophisticated means of purification.

“Subjects” (or “patients”) to be treated with the methods andcompositions described herein include both human subjects and animalsubjects (particularly other mammalian subjects such as dogs, cats,horses, monkeys, etc.) for veterinary purposes. Human subjects areparticularly preferred. The subjects may be male or female and may beany age, including neonate, infant, juvenile, adolescent, adult, andgeriatric subjects.

The disclosures of all United States patent references cited herein areto be incorporated herein by reference.

The ability of extracted keratin solutions to spontaneouslyself-assemble at the micron scale was published in two papers in 1986and 1987 (Thomas H et al., Int J Biol Macromol 1986; 8:258-64; van deLocht M, Melliand Textilberichte 1987; 10:780-6). This phenomenon is notsurprising given the highly controlled superstructure whence hairkeratins are obtained. When processed correctly, this ability toself-assemble can be preserved and used to create regular architectureson a size scale conducive to cellular infiltration. When keratins arehydrolyzed (e.g., with acids or bases), their molecular weight isreduced and they lose the ability to self-assemble. Therefore,processing conditions that minimize hydrolysis are preferred.

This ability to self-assemble is a particularly useful characteristicfor tissue engineering scaffolds for two reasons. First, self-assemblyresults in a highly regular structure with reproducible architectures,dimensionality, and porosity. Second, the fact that these architecturesform of their own accord under benign conditions allows for theincorporation of cells as the matrix is formed. These two features arecritically important to any system that attempts to mimic the nativeextracellular matrix (ECM).

Cellular recognition is also an important characteristic of biomaterialsthat seek to mimic the ECM. Such recognition is facilitated by thebinding of cell surface integrins to specific amino acid motifspresented by the constituent ECM proteins. Predominant proteins includecollagen and fibronectin, both of which have been extensively studiedwith regard to cell binding. Both proteins contain several regions thatsupport attachment by a wide variety of cell types. It has been shownthat in addition to the widely know Arginine-Glycine-Aspartic Acid (RGD)motif, the “X”-Aspartic Acid-“Y” motif on fibronectin is also recognizedby the integrin α4β1, where X equals Glycine, Leucine, or Glutamic Acid,and Y equals Serine or Valine. Keratin biomaterials derived from humanhair contain these same binding motifs. A search of the NCBI proteindatabase revealed sequences for 71 discrete, unique human hair keratinproteins. Of these, 55 are from the high molecular weight, low sulfur,alpha-helical family. This group of proteins is often referred to as thealpha-keratins and is responsible for imparting toughness to human hairfibers. These alpha-keratins have molecular weights greater than 40 kDaand an average cysteine (the main amino acid responsible for inter- andintramolecular protein bonding) content of 4.8 mole percent. Moreover,analysis of the amino acid sequences of these alpha keratin proteinsshowed that 78% contain at least one fibronectin-like integrin receptorbinding motif, and 25% contain at least two or more. Two recent papershave highlighted the fact that these binding sites are likely present onthe surface of keratin biomaterials by demonstrating excellent celladhesion onto processed keratin foams (Tachibana A et al., J Biotech2002; 93:165-70; Tachibana A et al., Biomaterials 2005; 26(3):297-302).

Other examples of natural polymers that may be utilized in a similarfashion to the disclosed keratin preparations include, but are notlimited to, collagen, gelatin, fibronectin, vitronectin, laminin,fibrin, mucin, elastin, nidogen (entactin), proteoglycans, etc. (See,e.g., U.S. Pat. No. 5,691,203 to Katsuen et al.).

There are two theories for the biological activity of human hairextracts. The first is that the human hair keratins (“HHKs”) themselvesare biologically active. Over 70 human hair keratins are known and theircDNA-derived sequences published. However, the full compliment of HHKsis unknown and estimates of over 100 have been proposed (Gillespie J M,The structural proteins of hair: isolation characterization, andregulation of biosynthesis. Goldsmith L A (editor), Biochemistry andphysiology of the skin (1983), Oxford University Press. New York;475-510). Within the complete range of HHKs are a small number that havebeen shown to participate in wound contracture and cell migration(Martin, P, Science 1997; 276:75-81). In particular, keratins K-6 andK-16 are expressed in the epidermis during wound healing and are alsofound in the outer root sheath of the hair follicle (Bowden P E,Molecular Aspects of Dermatology (1993), John Wiley & Sons, Inc.,Chichester: 19-54). The presence of these HHKs in extracts of humanhair, and their subsequent dosing directly into a wound bed, may beresponsible for “shortcutting” the otherwise lengthy process ofdifferentiation, migration, and proliferation, or for alleviating somebiochemical deficiency, thereby accelerating the tissue repair andregeneration process.

It has been known for more than a decade that growth factors such asbone morphogenetic protein-4 (BMP-4) and other members of thetransforming growth factor-β (TGF-β) superfamily are present indeveloping hair follicles (Jones C M et al., Development 1991;111:531-42; Lyons K M et al., Development 1990; 109:833-44; Blessings Met al., Genes and Develop 1993; 7:204-15). In fact, more than 30 growthfactors and cytokines are involved in the growth of a cycling hairfollicle (Hardy M H, Trends Genet 1992; 8(2):55-61; Stenn K S et al., JDermato Sci 1994; 7S:S109-24; Rogers G E, Int J Dev Biol 2004;48(2-3):163-70). Many of these molecules have a pivotal role in theregeneration of a variety of tissues. It is highly probable that anumber of growth factors become entrained within human hair whencytokines bind to stem cells residing in the bulge region of the hairfollicle (Panteleyev A A et al., J Cell Sci 2001; 114:3419-31). Thesegrowth factors would most certainly be extracted along with the keratinsfrom end-cut human hair. This observation is not without precedent, asit has previously been shown that many different types of growth factorsare present in the extracts of various tissues, and that their activityis maintained even after chemical extraction. Observations such as theseshow mounting evidence that a number of growth factors may be present inend-cut human hair, and that the keratins may be acting as a highlyeffective delivery matrix of, inter alia, these growth factors.

Keratins are a family of proteins found in the hair, skin, and othertissues of vertebrates. Hair is a unique source of human keratinsbecause it is one of the few human tissues that is readily available andinexpensive. Although other sources of keratins are acceptablefeedstocks for the present invention, (e.g. wool, fur, horns, hooves,beaks, feathers, scales, and the like), human hair is preferred for usewith human subjects because of its biocompatibility.

Keratins can be extracted from human hair fibers by oxidation orreduction using methods that have been published in the art (See, e.g.,Crewther W G et al. The chemistry of keratins, in Advances in proteinchemistry 1965; 20:191-346). These methods typically employ a two-stepprocess whereby the crosslinked structure of keratins is broken down byeither oxidation or reduction. In these reactions, the disulfide bondsin cysteine amino acid residues are cleaved, rendering the keratinssoluble (Scheme 1). The cuticle is essentially unaffected by thistreatment, so the majority of the keratins remain trapped within thecuticle's protective structure. In order to extract these keratins, asecond step using a denaturing solution must be employed. Alternatively,in the case of reduction reactions, these steps can be combined.Denaturing solutions known in the art include urea, transition metalhydroxides, surfactant solutions, and combinations thereof. Preferredmethods use aqueous solutions of tris in concentrations between 0.1 and1.0 M, and urea solutions between 0.1 and 10M, for oxidation andreduction reactions, respectively.

If one employs an oxidative treatment, the resulting keratins arereferred to as “keratoses.” If a reductive treatment is used, theresulting keratins are referred to as “kerateines” (See Scheme 1)

Crude extracts of keratins, regardless of redox state, can be furtherrefined into “gamma” and “alpha” fractions, e.g., by isoelectricprecipitation. High molecular weight keratins, or “alpha keratins,”(alpha helical), are thought to derive from the microfibrillar regionsof the hair follicle, and typically range in molecular weight from about40-85 kiloDaltons. Low molecular weight keratins, or “gamma keratins,”(globular), are thought to derive from the extracellular matrix regionsof the hair follicle, and typically range in molecular weight from about10-15 kiloDaltons. (See Crewther W G et al. The chemistry of keratins,in Advances in Protein Chemistry 1965; 20:191-346)

Even though alpha and gamma keratins possess unique properties, theproperties of subfamilies of both alpha and gamma keratins can only berevealed through more sophisticated means of purification. For example,keratins may be fractionated into “acidic” and “basic” proteinfractions. A preferred method of fractionation is ion exchangechromatography. These fractions possess unique properties, such as theirdifferential effects on blood cell aggregation (See Table 1 below; Seealso: U.S. Patent Application Publication No. 2006/0051732).

“Keratin derivative” as used herein refers to any keratin fractionation,derivative, subfamily, etc., or mixtures thereof, alone or incombination with other keratin derivatives or other ingredients,including but not limited to alpha keratose, gamma keratose, alphakerateine, gamma kerateine, meta keratin, keratin intermediatefilaments, and combinations thereof, including the acidic and basicconstituents thereof unless specified otherwise, along with variationsthereof that will be apparent to persons skilled in the art in view ofthe present disclosure. In some embodiments, the keratin derivativecomprises, consists or consists essentially of a particular fraction orsubfraction of keratin. The derivative may comprise, consist or consistessentially of at least 80, 90, 95 or 99 percent by weight of saidfraction or subfraction (or more).

In some embodiments, the keratin derivative comprises, consists of, orconsists essentially of acidic alpha keratose.

In some embodiments, the keratin derivative comprises, consists of orconsists essentially of alpha keratose, where the alpha keratosecomprises, consists of or consists essentially of at least 80, 90, 95 or99 percent by weight of acidic alpha keratose (or more), and where thealpha keratose comprises, consists of or consists essentially of notmore than 20, 10, 5 or 1 percent by weight of basic alpha keratose (orless).

In some embodiments, the keratin derivative comprises, consists of, orconsists essentially of basic alpha keratose.

In some embodiments, the keratin derivative comprises, consists of orconsists essentially of alpha keratose, where the alpha keratosecomprises, consists of or consists essentially of at least 80, 90, 95 or99 percent by weight of basic alpha keratose (or more), and where thealpha keratose comprises, consists of or consists essentially of notmore than 20, 10, 5 or 1 percent by weight of acidic alpha keratose (orless).

In some embodiments, the keratin derivative comprises, consists of, orconsists essentially of acidic alpha kerateine.

In some embodiments, the keratin derivative comprises, consists of orconsists essentially of alpha kerateine, where the alpha kerateinecomprises, consists of or consists essentially of at least 80, 90, 95 or99 percent by weight of acidic alpha kerateine (or more), and where thealpha kerateine comprises, consists of or consists essentially of notmore than 20, 10, 5 or 1 percent by weight of basic alpha kerateine (orless).

In some embodiments, the keratin derivative comprises, consists of, orconsists essentially of basic alpha kerateine.

In some embodiments, the keratin derivative comprises, consists of orconsists essentially of alpha kerateine, where the alpha kerateinecomprises, consists of or consists essentially of at least 80, 90, 95 or99 percent by weight of basic alpha kerateine (or more), and where thealpha kerateine comprises, consists of or consists essentially of notmore than 20, 10, 5 or 1 percent by weight of acidic alpha kerateine (orless).

In some embodiments, the keratin derivative comprises, consists of orconsists essentially of unfractionated alpha+gamma-kerateines. In someembodiments, the keratin derivative comprises, consists of or consistsessentially of acidic alpha+gamma-kerateines. In some embodiments, thekeratin derivative comprises, consists of or consists essentially ofbasic alpha+gamma-kerateines.

In some embodiments, the keratin derivative comprises, consists of orconsists essentially of unfractionated alpha+gamma-keratose. In someembodiments, the keratin derivative comprises, consists of or consistsessentially of acidic alpha+gamma-keratose. In some embodiments, thekeratin derivative comprises, consists of or consists essentially ofbasic alpha+gamma-keratose.

In some embodiments, the keratin derivative comprises, consists of orconsists essentially of unfractionated beta-keratose (e.g., derived fromcuticle). In some embodiments, the keratin derivative comprises,consists of or consists essentially of basic beta-keratose. In someembodiments, the keratin derivative comprises, consists of or consistsessentially of acidic beta-keratose.

The basic alpha keratose is preferably produced by separating basicalpha keratose from a mixture comprising acidic and basic alphakeratose, e.g., by ion exchange chromatography, and optionally the basicalpha keratose has an average molecular weight of from 10 to 100 or 200kiloDaltons. More preferably, the average molecular weight is from 30 or40 to 90 or 100 kiloDaltons. Optionally but preferably the processfurther comprises the steps of re-disolving said basic alpha-keratose ina denaturing and/or buffering solution, optionally in the presence of achelating agent to complex trace metals, and then re-precipitating thebasic alpha keratose from the denaturing solution. It will beappreciated that the composition preferably contains not more than 5, 2,1, or 0.1 percent by weight of acidic alpha keratose, or less.

The acidic alpha keratose is preferably produced by a reciprocal of theforegoing technique: that is, by separating and retaining acidic alphakeratose from a mixture of acidic and basic alpha keratose, e.g., by ionexchange chromatography, and optionally the acidic alpha keratose has anaverage molecular weight of from 10 to 100 or 200 kiloDaltons. Morepreferably, the average molecular weight is from 30 or 40 to 90 or 100kiloDaltons. Optionally but preferably the process further comprises thesteps of re-dissolving said acidic alpha-keratose in a denaturingsolution and/or buffering solution, optionally in the presence of achelating agent to complex trace metals, and then re-precipitating thebasic alpha keratose from the denaturing solution. It will beappreciated that the composition preferably contains not more than 5, 2,1, or 0.1 percent by weight of basic alpha keratose, or less.

Basic and acidic fractions of other keratoses can be prepared in likemanner as described above for basic and acidic alpha keratose.

The basic alpha kerateine is preferably produced by separating basicalpha kerateine from a mixture of acidic and basic alpha kerateine,e.g., by ion exchange chromatography, and optionally the basic alphakerateine has an average molecular weight of from 10 to 100 or 200kiloDaltons. More preferably, the average molecular weight is from 30 or40 to 90 or 100 kiloDaltons. Optionally but preferably the processfurther comprises the steps of re-dissolving said basic alpha-kerateinein a denaturing and/or buffering solution, optionally in the presence ofa chelating agent to complex trace metals, and then re-precipitating thebasic alpha kerateine from the denaturing solution. It will beappreciated that the composition preferably contains not more than 5, 2,1, or 0.1 percent by weight of acidic alpha kerateine, or less.

The acidic alpha kerateine is preferably produced by a reciprocal of theforegoing technique: that is, by separating and retaining acidic alphakerateine from a mixture of acidic and basic alpha kerateine, e.g., byion exchange chromatography, and optionally the acidic alpha kerateinehas an average molecular weight of from 10 to 100 or 200 kiloDaltons.Optionally but preferably the process further comprises the steps ofre-dissolving said acidic alpha-kerateine in a denaturing and/orbuffering solution), optionally in the presence of a chelating agent tocomplex trace metals, and then re-precipitating the basic alphakerateine from the denaturing solution. It will be appreciated that thecomposition preferably contains not more than 5, 2, 1, or 0.1 percent byweight of basic alpha kerateine, or less.

Basic and acidic fractions of other kerateines can be prepared in likemanner as described above for basic and acidic alpha kerateine.

Keratin materials are derived from any suitable source, including, butnot limited to, wool and human hair. In one embodiment keratin isderived from end-cut human hair, obtained from barbershops and salons.The material is washed in hot water and mild detergent, dried, andextracted with a nonpolar organic solvent (typically hexane or ether) toremove residual oil prior to use.

Keratoses. Keratose fractions are obtained by any suitable technique. Inone embodiment they are obtained using the method of Alexander andcoworkers (P. Alexander et al., Biochem. J 46, 27-32 (1950)). Basically,the hair is reacted with an aqueous solution of peracetic acid atconcentrations of less than ten percent at room temperature for 24hours. The solution is filtered and the alpha-keratose fractionprecipitated by addition of mineral acid to a pH of approximately 4. Thealpha-keratose is separated by filtration, washed with additional acid,followed by dehydration with alcohol, and then freeze dried. Increasedpurity can be achieved by redissolving the keratose in a denaturingsolution such as 7M urea, aqueous ammonium hydroxide solution, or 20 mMtris base buffer solution (e.g., Trizma® base), re-precipitating,re-dissolving, dialyzing against deionized water, and re-precipitatingat pH 4.

A preferred method for the production of keratoses is by oxidation withhydrogen peroxide, peracetic acid, or performic acid. A most preferredoxidant is peracetic acid. Preferred concentrations range from 1 to 10weight/volume percent (w/v %), the most preferred being approximately 2w/v %. Those skilled in the art will recognize that slight modificationsto the concentration can be made to effect varying degrees of oxidation,with concomitant alterations in reaction time, temperature, and liquidto solid ratio. It has also been discussed by Crewther et al. thatperformic acid offers the advantage of minimal peptide bond cleavagecompared to peracetic acid. However, peractic acid offers the advantagesof cost and availability. A preferred oxidation temperature is between 0and 100 degrees Celsius (° C.). A most preferred oxidation temperatureis 37° C. A preferred oxidation time is between 0.5 and 24 hours. A mostpreferred oxidation time is 12 hours. A preferred liquid to solid ratiois from 5 to 100:1. A most preferred ratio is 20:1. After oxidation, thehair is rinsed free of residual oxidant using a copious amount ofdistilled water.

The keratoses can be extracted from the oxidized hair using an aqueoussolution of a denaturing agent. Protein denaturants are well known inthe art, but preferred solutions include urea, transition metalhydroxides (e.g. sodium and potassium hydroxide), ammonium hydroxide,and tris(hydroxymethyl)aminomethane (tris base). A preferred solution isTrizma® base (a brand of tris base) in the concentration range from 0.01to 1M. A most preferred concentration is 0.1M. Those skilled in the artwill recognize that slight modifications to the concentration can bemade to effect varying degrees of extraction, with concomitantalterations in reaction time, temperature, and liquid to solid ratio. Apreferred extraction temperature is between 0 and 100 degrees Celsius. Amost preferred extraction temperature is 37° C. A preferred extractiontime is between 0.5 and 24 hours. A most preferred extraction time is 3hours. A preferred liquid to solid ratio is from 5 to 100:1. A mostpreferred ratio is 40:1. Additional yield can be achieved withsubsequent extractions with dilute solutions of tris base or deionized(DI) water. After extraction, the residual solids are removed fromsolution by centrifugation and/or filtration.

The crude extract can be isolated by first neutralizing the solution toa pH between 7.0 and 7.4. A most preferred pH is 7.4. Residualdenaturing agent is removed by dialysis against DI water. Concentrationof the dialysis retentate is followed by lyophilization or spray drying,resulting in a dry powder mixture of both gamma- and alpha-keratose.Alternately, alpha-keratose is isolated from the extract solution bydropwise addition of acid until the pH of the solution reachesapproximately 4.2. Preferred acids include sulfuric, hydrochloric, andacetic. A most preferred acid is concentrated hydrochloric acid.Precipitation of the alpha fraction begins at around pH 6.0 andcontinues until approximately 4.2. Fractional precipitation can beutilized to isolate different ranges of protein with differentisoelectric properties. Solid alpha-keratose can be recovered bycentrifugation or filtration.

The alpha keratose can be further purified by re-dissolving the solidsin a denaturing solution. The same denaturing solutions as thoseutilized for extraction can be used, however a preferred denaturingsolution is tris base. Ethylene diamine tetraacetic acid (EDTA) can beadded to complex and remove trace metals found in the hair. A preferreddenaturing solution is 20 mM tris base with 20 mM EDTA or DI water with20 mM EDTA. If the presence of trace metals is not detrimental to theintended application, the EDTA can be omitted. The alpha-keratose isre-precipitated from this solution by dropwise addition of hydrochloricacid to a final pH of approximately 4.2. Isolation of the solid is bycentrifugation or filtration. This process can be repeated several timesto further purify the alpha-keratose.

The gamma keratose fraction remains in solution at pH 4 and is isolatedby addition to a water-miscible organic solvent such as alcohol,followed by filtration, dehydrated with additional alcohol, and freezedried. Increased purity can be achieved by re-dissolving the keratose ina denaturing solution such as 7M urea, aqueous ammonium hydroxidesolution, or 20 mM tris buffer solution, reducing the pH to 4 byaddition of a mineral acid, removing any solids that form, neutralizingthe supernatant, re-precipitating the protein with alcohol,re-dissolving, dialyzing against deionized water, and re-precipitatingby addition to alcohol. The amount of alcohol consumed in these stepscan be minimized by first concentrating the keratose solution bydistillation.

After removal of the alpha keratose, the concentration of gamma keratosefrom a typical extraction solution is approximately 1-2%. The gammakeratose fraction can be isolated by addition to a water-misciblenon-solvent. To effect precipitation, the gamma-keratose solution can beconcentrated by evaporation of excess water. This solution can beconcentrated to approximately 10-20% by removal of 90% of the water.This can be done using vacuum distillation or by falling filmevaporation. After concentration, the gamma-keratose solution is addeddropwise to an excess of cold non-solvent. Suitable non-solvents includeethanol, methanol, acetone, and the like. A most preferred non-solventis ethanol. A most preferred method is to concentrate the gamma keratosesolution to approximately 10 w/v % protein and add it dropwise to an8-fold excess of cold ethanol. The precipitated gamma keratose can beisolated by centrifugation or filtration and dried. Suitable methods fordrying include freeze drying (lyophilization), air drying, vacuumdrying, or spray drying. A most preferred method is freeze drying.

Kerateines. Kerateine fractions can be obtained using a combination ofthe methods of Bradbury and Chapman (J. Bradbury et al., Aust. J. Biol.Sci. 17, 960-72 (1964)) and Goddard and Michaelis (D. Goddard et al., J.Biol. Chem. 106, 605-14 (1934)). Essentially, the cuticle of the hairfibers is removed ultrasonically in order to avoid excessive hydrolysisand allow efficient reduction of cortical disulfide bonds in a secondstep. The hair is placed in a solution of dichloroacetic acid andsubjected to treatment with an ultrasonic probe. Further refinements ofthis method indicate that conditions using 80% dichloroacetic acid,solid to liquid of 1:16, and an ultrasonic power of 180 Watts areoptimal (H. Ando et al., Sen'i Gakkaishi 31(3), T81-85 (1975)). Solidfragments are removed from solution by filtration, rinsed and air dried,followed by sieving to isolate the hair fibers from removed cuticlecells.

In some embodiments, following ultrasonic removal of the cuticle, alpha-and gamma-kerateines are obtained by reaction of the denuded fibers withmercaptoethanol. Specifically, a low hydrolysis method is used at acidicpH (E. Thompson et al., Aust. J. Biol. Sci. 15, 757-68 (1962)). In atypical reaction, hair is extracted for 24 hours with 4M mercaptoethanolthat has been adjusted to pH 5 by addition of a small amount ofpotassium hydroxide in deoxygenated water containing 0.02M acetatebuffer and 0.001 M surfactant.

The solution is filtered and the alpha-kerateine fraction precipitatedby addition of mineral acid to a pH of approximately 4. Thealpha-kerateine is separated by filtration, washed with additional acid,followed by dehydration with alcohol, and then dried under vacuum.Increased purity is achieved by re-dissolving the kerateine in adenaturing solution such as 7M urea, aqueous ammonium hydroxidesolution, or 20 mM tris buffer solution, re-precipitating,re-dissolving, dialyzing against deionized water, and re-precipitatingat pH 4.

The gamma kerateine fraction remains in solution at pH 4 and is isolatedby addition to a water-miscible organic solvent such as alcohol,followed by filtration, dehydrated with additional alcohol, and driedunder vacuum. Increased purity can be achieved by re-dissolving thekerateine in a denaturing solution such as 7M urea, aqueous ammoniumhydroxide solution, or 20 mM tris buffer solution, reducing the pH to 4by addition of a mineral acid, removing any solids that form,neutralizing the supernatant, re-precipitating the protein with alcohol,re-dissolving, dialyzing against deionized water, and reprecipitating byaddition to alcohol. The amount of alcohol consumed in these steps canbe minimized by first concentrating the keratin solution bydistillation.

In an alternate method, the kerateine fractions are obtained by reactingthe hair with an aqueous solution of sodium thioglycolate.

A preferred method for the production of kerateines is by reduction ofthe hair with thioglycolic acid or beta-mercaptoethanol. A mostpreferred reductant is thioglycolic acid (TGA). Preferred concentrationsrange from 1 to 10M, the most preferred being approximately 1.0M. Thoseskilled in the art will recognize that slight modifications to theconcentration can be made to effect varying degrees of reduction, withconcomitant alterations in pH, reaction time, temperature, and liquid tosolid ratio. A preferred pH is between 9 and 11. A most preferred pH is10.2. The pH of the reduction solution is altered by addition of base.Preferred bases include transition metal hydroxides, sodium hydroxide,and ammonium hydroxide. A most preferred base is sodium hydroxide. ThepH adjustment is effected by dropwise addition of a saturated solutionof sodium hydroxide in water to the reductant solution. A preferredreduction temperature is between 0 and 100° C. A most preferredreduction temperature is 37° C. A preferred reduction time is between0.5 and 24 hours. A most preferred reduction time is 12 hours. Apreferred liquid to solid ratio is from 5 to 100:1. A most preferredratio is 20:1. Unlike the previously described oxidation reaction,reduction is carried out at basic pH. That being the case, keratins arehighly soluble in the reduction media and are expected to be extracted.The reduction solution is therefore combined with the subsequentextraction solutions and processed accordingly.

Reduced keratins are not as hydrophilic as their oxidized counterparts.As such, reduced hair fibers will not swell and split open as willoxidized hair, resulting in relatively lower yields. Another factoraffecting the kinetics of the reduction/extraction process is therelative solubility of kerateines. The relative solubility rankings inwater is gamma-keratose>alpha-keratose>gamma-kerateine>alpha-kerateinefrom most to least soluble. Consequently, extraction yields from reducedhair fibers are not as high. This being the case, subsequent extractionsare conducted with additional reductant plus denaturing agent solutions.Preferred solutions for subsequent extractions include TGA plus urea,TGA plus tris base, or TGA plus sodium hydroxide. After extraction,crude fractions of alpha- and gamma-kerateine can be isolated using theprocedures described for keratoses. However, precipitates of gamma- andalpha-kerateine re-form their cystine crosslinks upon exposure tooxygen. Precipitates must therefore be re-dissolved quickly to avoidinsolubility during the purification stages, or precipitated in theabsence of oxygen.

Residual reductant and denaturing agents can be removed from solution bydialysis. Typical dialysis conditions are 1 to 2% solution of kerateinesdialyzed against DI water for 24 to 72 hours. Those skilled in the artwill recognize that other methods exist for the removal of low molecularweight contaminants in addition to dialysis (e.g. microfiltration,chromatography, and the like). The use of tris base is only required forinitial solubilization of the kerateines. Once dissolved, the kerateinesare stable in solution without the denaturing agent. Therefore, thedenaturing agent can be removed without the resultant precipitation ofkerateines, so long as the pH remains at or above neutrality. The finalconcentration of kerateines in these purified solutions can be adjustedby the addition/removal of water.

Regardless of the form of the keratin (i.e. keratoses or kerateines),several different approaches to further purification can be employed tokeratin solutions. Care must be taken, however, to choose techniquesthat lend themselves to keratin's unique solubility characteristics. Oneof the most simple separation technologies is isoelectric precipitation.In this method, proteins of differing isoelectric point can be isolatedby adjusting the pH of the solution and removing the precipitatedmaterial. In the case of keratins, both gamma- and alpha-forms aresoluble at pH>6.0. As the pH falls below 6, however, alpha-keratinsbegin to precipitate. Keratin fractions can be isolated by stopping theprecipitation at a given pH and separating the precipitate bycentrifugation and/or filtration. At a pH of approximately 4.2,essentially all of the alpha-keratin will have been precipitated. Theseseparate fractions can be re-dissolved in water at neutral pH, dialyzed,concentrated, and reduced to powders by lyophilization or spray drying.However, kerateine fractions must be stored in the absence of oxygen orin dilute solution to avoid crosslinking.

Another general method for separating keratins is by chromatography.Several types of chromatography can be employed to fractionate keratinsolutions including size exclusion or gel filtration chromatography,affinity chromatography, isoelectric focusing, gel electrophoresis, ionexchange chromatography, and immunoaffinity chromatography. Thesetechniques are well known in the art and are capable of separatingcompounds, including proteins, by the characteristics of molecularweight, chemical functionality, isoelectric point, charge, orinteractions with specific antibodies, and can be used alone or in anycombination to effect high degrees of separation and resulting purity.

A preferred purification method is ion exchange (IEx) chromatography.IEx chromatography is particularly suited to protein separation owningto the amphiphilic nature of proteins in general and keratins inparticular. Depending on the starting pH of the solution, and thedesired fraction slated for retention, either cationic or anionic IEx(CIEx or AIEx, respectively) techniques can be used. For example, at apH of 6 and above, both gamma- and alpha-keratins are soluble and abovetheir isoelectric points. As such, they are anionic and can be bound toan anionic exchange resin. However, it has been discovered that asub-fraction of keratins does not bind to a weakly anionic exchangeresin and instead passes through a column packed with such resin. Apreferred solution for AIEx chromatography is purified or fractionatedkeratin, isolated as described previously, in purified water at aconcentration between 0 and 5 weight/volume %. A preferred concentrationis between 0 and 4 w/v %. A most preferred concentration isapproximately 2 w/v %. It is preferred to keep the ionic strength ofsaid solution initially quite low to facilitate binding to the AIExcolumn. This is achieved by using a minimal amount of acid to titrate apurified water solution of the keratin to between pH 6 and 7. A mostpreferred pH is 6. This solution can be loaded onto an AIEx column suchas DEAE-Sepharose® resin or Q-Sepharose® resin columns. A preferredcolumn resin is DEAE-Sepharose® resin. The solution that passes throughthe column can be collected and further processed as describedpreviously to isolate a fraction of acidic keratin powder.

In some embodiments the activity of the keratin matrix is enhanced byusing an AIEx column to produce the keratin that may be useful for,inter alia, promoting cell adhesion. Without wishing to be bound to anyparticular theory, it is envisioned that the fraction that passesthrough an anionic column, i.e. acidic keratin, promotes cell adhesion.

Another fraction binds readily, and can be washed off the column usingsalting techniques known in the art. A preferred elution medium issodium chloride solution. A preferred concentration of sodium chlorideis between 0.1 and 2M. A most preferred concentration is 2M. The pH ofthe solution is preferred to be between 6 and 12. A most preferred pH is12. In order to maintain stable pH during the elution process, a buffersalt can be added. A preferred buffer salt is Trizma® base. Thoseskilled in the art will recognize that slight modifications to the saltconcentration and pH can be made to effect the elution of keratinfractions with differing properties. It is also possible to usedifferent salt concentrations and pH's in sequence, or employ the use ofsalt and/or pH gradients to produce different fractions. Regardless ofthe approach taken, however, the column eluent can be collected andfurther processed as described previously to isolate fractions of basickeratin powders.

A complimentary procedure is also feasible using CIEx techniques.Namely, the keratin solution can be added to a cation exchange resinsuch as SP Sepharose® resin (strongly cationic) or CM Sepharose® resin(weakly cationic), and the basic fraction collected with the passthrough. The retained acid keratin fraction can be isolated by saltingas previously described.

Meta Keratins. Meta keratins are synthesized from both the alpha andgamma fractions of kerateine using substantially the same procedures.Basically, the kerateine is dissolved in a denaturing solution such as7M urea, aqueous ammonium hydroxide solution, or 20 mM tris buffersolution. Pure oxygen is bubbled through the solution to initiateoxidative coupling reactions of cysteine groups. The progress of thereaction is monitored by an increase in molecular weight as measuredusing SDS-PAGE. Oxygen is continually bubbled through the reactionsolution until a doubling or tripling of molecular weight is achieved.The pH of the denaturing solution can be adjusted to neutrality to avoidhydrolysis of the proteins by addition of mineral acid.

Keratin Intermediate Filaments. IFs of human hair fibers are obtainedusing the method of Thomas and coworkers (H. Thomas et al., Int. J.Biol. Macromol. 8, 258-64 (1986)). This is essentially a chemicaletching method that reacts away the keratin matrix that serves to “glue”the IFs in place, thereby leaving the IFs behind. In a typicalextraction process, swelling of the cuticle and sulfitolysis of matrixproteins is achieved using 0.2M Na₂SO₃, 0.1M Na₂O₆S₄ in 8M urea and 0.1MTris-HCl buffer at pH 9. The extraction proceeds at room temperature for24 hours. After concentrating, the dissolved matrix keratins and IFs areprecipitated by addition of zinc acetate solution to a pH ofapproximately 6. The IFs are then separated from the matrix keratins bydialysis against 0.05M tetraborate solution. Increased purity isobtained by precipitating the dialyzed solution with zinc acetate,redissolving the IFs in sodium citrate, dialyzing against distilledwater, and then freeze drying the sample.

Further discussion of keratin preparations are found in U.S. PatentApplication Publication 2006/0051732 (Van Dyke), which is incorporatedby reference herein.

Formulations. Dry powders may be formed of keratin derivatives asdescribed above in accordance with known techniques such as freezedrying (lyophilization). In some embodiments, compositions of theinvention may be produced by mixing such a dry powder composition formwith an aqueous solution to produce a composition comprising anelectrolyte solution having said keratin derivative solubilized therein.The mixing step can be carried out at any suitable temperature,typically room temperature, and can be carried out by any suitabletechnique such as stirring, shaking, agitation, etc. The salts and otherconstituent ingredients of the electrolyte solution (e.g., allingredients except the keratin derivative and the water) may becontained entirely in the dry powder, entirely within the aqueouscomposition, or may be distributed between the dry powder and theaqueous composition. For example, in some embodiments, at least aportion of the constituents of the electrolyte solution is contained inthe dry powder.

The formation of a matrix comprising keratin materials such as describedabove can be carried out in accordance with techniques long establishedin the field or variations thereof that will be apparent to thoseskilled in the art. In some embodiments, the keratin preparation isdried and rehydrated prior to use. See, e.g., U.S. Pat. No. 2,413,983 toLustig et al., U.S. Pat. No. 2,236,921 to Schollkipf et al., and U.S.Pat. No. 3,464,825 to Anker. In preferred embodiments, the matrix, orhydrogel, is formed by re-hydration of the lyophilized material with asuitable solvent, such as water or phosphate buffered saline (PBS). Thegel can be sterilized, e.g., by γ-irradiation (800 krad) using a Co60source. Other suitable methods of forming keratin matrices include, butare not limited to, those found in U.S. Pat. No. 6,270,793 (Van Dyke etal.), U.S. Pat. No. 6,274,155 (Van Dyke et al.), U.S. Pat. No. 6,316,598(Van Dyke et al.), U.S. Pat. No. 6,461,628 (Blanchard et al.), U.S. Pat.No. 6,544,548 (Siller-Jackson et al.), and U.S. Pat. No. 7,01,987 (VanDyke).

In some composition embodiments, the keratin derivatives (particularlyalpha and/or gamma kerateine and alpha and/or gamma keratose) have anaverage molecular weight of from about 10 to 70 or 85 or 100kiloDaltons. Other keratin derivatives, particularly meta-keratins, mayhave higher average molecular weights, e.g., up to 200 or 300kiloDaltons. In general, the keratin derivative (this term includingcombinations of derivatives) may be included in the composition in anamount of from about 0.1, 0.5 or 1 percent by weight up to 3, 4, 5, or10 percent by weight. The composition when mixed preferably has aviscosity of about 1 or 1.5 to 4, 8, 10 or 20 centipoise. Viscosity atany concentration can be modulated by changing the ratio of alpha togamma keratose.

The keratin derivative composition or formulation may optionally containone or more active ingredients such as one or more growth factors (e.g.,in an amount ranging from 0.0000001 to 1 or 5 percent by weight of thecomposition that comprises the keratin derivative(s)) to facilitategrowth or healing, facilitate or inhibit coagulation, facilitate orinhibit cell or tissue adhesion, etc. Examples of suitable activeingredients include but are not limited to nerve growth factor, vascularendothelial growth factor, fibronectin, fibrin, laminin, acidic andbasic fibroblast growth factors, testosterone, ganglioside GM-1,catalase, insulin-like growth factor-I (IGF-I), platelet-derived growthfactor (PDGF), neuronal growth factor galectin-1, and combinationsthereof. See, e.g., U.S. Pat. No. 6,506,727 to Hansson et al. and U.S.Pat. No. 6,890,531 to Horie et al.

As used herein, “growth factors” include molecules that promote theregeneration, growth and survival of tissue. Growth factors that areused in some embodiments of the present invention may be those naturallyfound in keratin extracts, or may be in the form of an additive, addedto the keratin extracts or formed keratin matrices. Examples of growthfactors include, but are not limited to, nerve growth factor (NGF) andother neurotrophins, platelet-derived growth factor (PDGF),erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growthdifferentiation factor-9 (GDF9), basic fibroblast growth factor (bFGF orFGF2), epidermal growth factor (EGF), hepatocyte growth factor (HGF),granulocyte-colony stimulating factor (G-CSF), andgranulocyte-macrophage colony stimulating factor (GM-CSF). There aremany structurally and evolutionarily related proteins that make up largefamilies of growth factors, and there are numerous growth factorfamilies, e.g., the neurotrophins (NGF, BDNF, and NT3). Theneurotrophins are a family of molecules that promote the growth andsurvival of, inter alia, nervous tissue. Examples of neurotrophinsinclude, but are not limited to, nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), andneurotrophin 4 (NT-4). See U.S. Pat. No. 5,843,914 to Johnson, Jr. etal.; U.S. Pat. No. 5,488,099 to Persson et al.; U.S. Pat. No. 5,438,121to Barde et al.; U.S. Pat. No. 5,235,043 to Collins et al.; and U.S.Pat. No. 6,005,081 to Burton et al.

For example, nerve growth factor (NGF) can be added to the keratinmatrix composition in an amount effective to promote the regeneration,growth and survival of various tissues. The NGF is provided inconcentrations ranging from 0.1 ng/mL to 1000 ng/mL. More preferably,NGF is provided in concentrations ranging from 1 ng/mL to 100 ng/mL, andmost preferably 10 ng/mL to 100 ng/mL. See U.S. Pat. No. 6,063,757 toUrso.

Other examples of natural polymers that may be prepared and utilized ina similar fashion to the disclosed keratin preparations include, but arenot limited to, collagen, gelatin, fibronectin, vitronectin and laminin(See, e.g., U.S. Pat. No. 5,691,203 to Katsuen et al.), with thenecessary modifications apparent to those skilled in the art.

The composition is preferably sterile and non-pryogenic. The compositionmay be provided preformed and aseptically packaged in a suitablecontainer, such as a flexible polymeric bag or bottle, or a foilcontainer, or may be provided as a kit of sterile dry powder in onecontainer and sterile aqueous solution in a separate container formixing just prior to use. When provided pre-formed and packaged in asterile container the composition preferably has a shelf life of atleast 4 or 6 months (up to 2 or 3 years or more) at room temperature,prior to substantial loss of viscosity (e.g., more than 10 or 20percent) and/or substantial precipitation of the keratin derivative(e.g., settling detectable upon visual inspection).

Coatings and Biomedical Implants. As noted above, the present inventionprovides an implantable biomedical device, comprising: a substrate and akeratin derivative on the substrate, wherein the keratin derivative ispresent in an amount effective to reduce cell and/or tissue adhesion tothe substrate. In some embodiments the keratin derivative comprises,consists of or consists essentially of basic alpha keratose, basic alphakerateine, or combinations thereof.

The chemistry of keratins can be utilized to optimize the properties ofkeratin-based coatings. Alpha and gamma keratoses have inert sulfurresidues. The oxidation reaction is a terminal step and results in theconversion of cystine residues into two non-reactive sulfonic acidresidues. Kerateines, on the other hand, have labile sulfur residues.During the creation of the kerateines, cystine is converted to cysteine,which can be a source of further chemical modifications (See Scheme 1).One such useful reaction is oxidative sulfur-sulfur coupling. Thisreaction simply converts the cysteine back to cystine and reforms thecrosslinks between proteins. This is a useful reaction for increasingthe molecular weight of the gamma or alpha fraction of interest, whichin turn will modify the bulk properties of the material. Increasingmolecular weight influences material properties such as viscosity, dryfilm strength, gel strength, etc. Such reformed kerateines are referredto as meta keratins.

Meta keratins can be derived from the gamma or alpha fractions, or acombination of both. Oxidative re-crosslinking of the kerateines isaffected by addition of an oxidizing agent such as peracetic acid orhydrogen peroxide. A preferred oxidizing agent is oxygen. This reactioncan be accomplished simply by bubbling oxygen through the kerateinesolution or by otherwise exposing the sample to air. Optimizing themolecular weight through the use of meta-keratins allows formulations tobe optimized for a variety of properties including viscosity, filmstrength and elasticity, fiber strength, and hydrolytic susceptibility.Crosslinking in air works to improve biocompatibility by providingbiomaterial with a minimum of foreign ingredients.

Any suitable substrate (typically a device intended for implanting intoor inserting into a human or animal subject) may be coated or treatedwith keratin materials or keratin derivatives as described herein,including but not limited to grafts such as vascular grafts, vascularstents, catheters, leads, pacemakers, cardioverters, valves, fastenersor ports such as heart valves, etc.

The substrate may be formed from any suitable material, including butnot limited to organic polymers (including stable polymers andbiodegradable or bioerodable polymers), natural materials (e.g.,collagen), metals (e.g., platinum, gold, stainless steel, etc.)inorganic materials such as silicon, glass, etc., and compositesthereof.

Coating of the substrate may be carried out by any suitable means, suchas spray coating, dip coating, or the like. In some embodiments, stepsmay be taken to couple or covalently couple the keratin to thesubstratem such as with a silane coupling agent, if so desired. Thekeratin derivative may be subsequently coated with another material,and/or other materials may be co-deposited with the keratin derivative,such as one or more additional active agents, stabilizers, coatings,etc.

Another aspect of the present invention is an implantable anti-adhesivetissue barrier, comprising: a solid, physiologically acceptablesubstrate (typically a sheet material, including but not limited tofilms, and woven and non-woven sheet materials formed from organicpolymers or natural materials); and a keratin derivative on thesubstrate. In some embodiments the keratin derivative comprises,consists of or consists essentially of basic alpha keratose, basic alphakerateine, or combinations thereof.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLE 1 Crude Keratose Samples

Keratose fractions were obtained using a method based on that ofAlexander and coworkers. However, the method was substantially modifiedto minimize hydrolysis of peptide bonds. Briefly, 50 grams of clean, dryhair that was collected from a local barber shop was reacted with 1000mL of an aqueous solution of 2 w/v % peracetic acid (PAA) at roomtemperature for 12 hr. The oxidized hair was recovered using a 500micron sieve, rinsed with copious amounts of DI water, and the excesswater removed. Keratoses were extracted from the oxidized hair fiberswith 1000 mL of 100 mM Trizma® base. After 3 hours, the hair wasseparated by sieve and the liquid neutralized by dropwise addition ofhydrochloric acid (HCl). Additional keratoses were extracted from theremaining hair with two subsequent extractions using 1000 mL of 0.1 MTrizma® base and 1000 mL of DI water, respectively. Each time the hairwas separated by sieve and the liquid neutralized with HCl. All threeextracts were combined, centrifuged, and any residual solid materialremoved by filtration. The combined extract was purified by tangentialflow dialysis against DI water with a 1 KDa nominal low molecular weightcutoff membrane. The solution was concentrated and lyophilized toproduce a crude keratose powder.

EXAMPLE 2 Crude Kerateine Samples

Kerateine fractions were obtained using a modification of the methoddescribed by Goddard and Michaelis. Briefly, the hair was reacted withan aqueous solution of 1M TGA at 37° C. for 24 hours. The pH of the TGAsolution had been adjusted to pH 10.2 by dropwise addition of saturatedNaOH solution. The extract solution was filtered to remove the reducedhair fibers and retained. Additional keratin was extracted from thefibers by sequential extractions with 1000 mL of 100 mM TGA at pH 10.2for 24 hours, 1000 mL of 10 mM TGA at pH 10.2 for 24 hours, and DI waterat pH 10.2 for 24 hours. After each extraction, the solution wascentrifuged, filtered, and added to the dialysis system. Eventually, allthe extracts were combined and dialyzed against DI water with a 1 KDanominal low molecular weight cutoff membrane. The solution wasconcentrated, titrated to pH 7, and stored at approximately 5% totalprotein concentration at 4° C. Alternately, the concentrated solutioncould be lyophilized and stored frozen and under nitrogen.

EXAMPLE 3 Ion Exchange Chromatography

Just prior to fractionation, keratose samples were re-dissolved inultrapure water and titrated to pH 6 by addition of dilute HCl solution.Kerateine samples were titrated to pH 6 by careful addition of diluteHCl solution as well. The samples were loaded onto a 200 mL flashchromatography column containing either DEAE-Sepharose (weakly anionic)or Q-Sepharose (strongly anionic) exchange resin (50-100 mesh;Sigma-Aldrich, Milwaukee, Wis.) with gentle pressure and the flowthrough collected (acidic keratin). A small volume of 10 mM Trizma® base(approximately 200 mL) at pH 6 was used to completely wash through thesample. Basic keratin was eluted from the column with 100 mM tris baseplus 2M NaCl at pH 12. Each sample was separately neutralized anddialyzed against DI water using tangential flow dialysis with a LMWCO of1 KDa, concentrated by rotary evaporation, and freeze dried.

EXAMPLE 4 Evaluation of Viscosity and Red Blood Cell Aggregation

As previously described, a sample of alpha-keratose was produced,separated on a DEAE-Sepharose IEx column into acidic and basicfractions, dissolved in PBS, and the pH adjusted to 7.4. These solutionswere prepared at 5 weight percent concentration and their RBCaggregation characteristics grossly evaluated with fresh whole humanblood by mixing at a 1:1 ratio. Samples were taken after 20 minutes andevaluated by light microscopy. The ion exchange chromatography washighly effective at separating the aggregation phenomenon (data notshown). Basic alpha-keratose was essentially free from interactions withblood cells while the acidic alpha-keratose caused excessiveaggregation.

Samples of acidic and basic alpha keratose, unfractionatedalpha+gamma-kerateines, unfractionated alpha+gamma-keratose, andbeta-keratose (derived from cuticle) were prepared at approximately 4w/v % and pH 7.4 in phosphate buffered saline (PBS). Samples were testedfor viscosity and red blood cell (RBC) aggregation. These results areshown in Table 1:

TABLE 1 Results of viscosity and RBC aggregation tests on keratinsolutions. Fluid formulations were prepared at approximately 4 w/v % inPBS at pH 7.4 and tested with human whole blood at a ratio of 1:1.Viscosity RBC Sample Description (centipoise) Aggregation* acidicalpha-keratose (1X AIEx) 5.65 3 acidic alpha-keratose (2X AIEx) 19.7 5basic alpha-keratose 1.57 2 alpha + gamma-keratose (hydrolyzed) 1.12 1alpha + gamma-kerateine (unfractionated) 1.59 2 *Degree of aggregation:1 = none, 5 = high

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of producing a charged fraction ofkeratin, comprising: providing a solution comprising acidic and basickeratin, wherein said solution has a pH of between 6 and 7, and whereinsaid solution is provided by: reacting human hair with thioglycolicacid, and extracting said acidic and basic keratin with a solutioncomprising tris base; separating a fraction of said acidic keratin or afraction of said basic keratin from said solution comprising said acidicand basic keratin, wherein said separating is carried out bychromatography; and collecting said fraction; to produce the chargedfraction of keratin.
 2. The method of claim 1, wherein said keratincomprises alpha keratin.
 3. The method of claim 1, wherein said keratincomprises gamma keratin.
 4. A method of producing a charged fraction ofalpha keratin, comprising: providing a solution comprising keratin,wherein said solution is provided by: reacting human hair withthioglycolic acid, and extracting said keratin with a solutioncomprising tris base; precipitating alpha keratin from said solutioncomprising keratin by addition of mineral acid to a pH of approximately4; separating precipitated alpha keratin from said solution comprisingkeratin; collecting the precipitated alpha keratin; and then,optionally, re-dissolving the alpha keratin at pH 9 and re-precipitatingat pH 4 to further purify; and then, re-dissolving the alpha keratin toprovide a purified alpha keratin solution and adjusting the pH of thepurified alpha keratin solution to be between 6 and 7,wherein thepurified alpha keratin solution comprises acidic and basic alphakeratin; and then, separating a fraction of said acidic alpha keratin ora fraction of said basic alpha keratin from said purified alpha keratinsolution, wherein said separating is carried out by chromatography; andcollecting said fraction; to thereby produce the charged fraction ofalpha keratin.
 5. The method of claim 1, wherein said keratin comprisesat least 80% by weight of alpha keratin.
 6. The method of claim 1,wherein said keratin comprises at least 90% by weight of alpha keratin.